Hepatoma Cells Suppress Anoikis Through Synoikis-like Mechanism

Cell-cell or cell-matrix contacts not merely provide structural anchorage for the cell but mediate pivotal survival signals to the cells. Disturbance of cell-anchorage will frequently lead to the immediate initiation of a suicide program—apoptosis, within the cell. The subset of apoptosis triggered by inadequate or inappropriate cell-matrix contacts was termed anoikis. Anoikis seems to play an important role in the physiological induction of apoptosis during development and maintenance of tissue homeostasis in the organism. Understanding anoikis seems to be of particular importance for cancer research as malignant cells, once they begin to metastasize, have obviously acquired properties rendering them resistant to death induced by loss of anchorage—i.e. a reable to detach from the primary tumor without undergoing apoptosis. Resistance to anoikis is the prerequisite of cancer cells metastasis.Elucidation of the mechanism of anoikis resistance remains a significant challenge. Tumor cells could resist to anoikis by a survival style named Synoikis, which was characterized by multi-cell aggregation formation and continuous proliferation. In order to study the mechanism of hepatoma cells anoikis resistance, we established a non-anchored culture model to investigate the response of hepatoma cells to their loss of anchorage. Our results showed that hepatoma cells could resist to anoikis by a process we called Synoikis-like which is characterized by rounded up and aggregated morphology change, proliferation block and cell cycle arrest. These suspended Synoikis-like cells could spontaneously attach again under suitable condition which indicate a reversible property of these suspended cells. Synoikis-like hepatoma cells were not only resistant to anoikis, but also resistant to extracellular stimuli, such as chemotherapy, apoptotic stimuli, and ultraviolet rays (UV) radiation. Microarray analysis showed that the physiology process of these Synoikis-like cells are significantly different from their attached predecessor (P=0. 0023). To further clarify the molecular mechanism involved in the anoikis resistant process of hepatoma cells, we select several candidate genes that may play key roles in this process for further investigation. These studies suggest that apoptosis regulatory pathway, BDNF/TrkB pathway, BCL2L2 may play a significant role in the anoikis resistance process of hepatoma cells.OBJECTIVES1) Establish a model to culture hepatoma cells in suspension.2) The process of hepatoma cells anoikis resistance was analyzed according to their biology features.3) To study the changes of cell signaling pathway when hepatoma cells gain ability to resist to anoikis.4) To study the expression profile of hepatoma cells while anoikis resistance happens, and screen the key molecules during the anoikis resistance process of hepatoma cells.5) To study the biological role of the key molecules in anoikis resistance.METHODS1 Construct a detached tissue culture model1.1 Establish a detached model and compare the morphological changes between attached and detached hepatoma cells 1.1.1 Establish a model to culture hepatoma cells in suspensionIn order to study the mechanisms of hepatoma cells anoikis resistance, PolyHEMA was prepared by dissolving in 95% ethanol to a concentration of 12 mg/ml. Coated plate with 2 mg/cm~2 of polyHEMA and allowed to air dry in a sterile environment.1.1.2 Dynamically observe the procession of hepatoma cells after they were detachedThe BEL7402 hepatoma cells were cultured in the polyHEMA plate, the progress of anoikis resistance and morphological changes were recorded. The viability of hepatoma cells were determined by trypan blue assay.1.1.3 Morphological changes of hepatoma cellsCalcium deprivation assay by EGTA was used to study the adhesion feature of Synoikis-like cells. The morphological changes were detected by Acridine Orange Fluorescence Staining and Heochest33342 staining. The ultrastructure of Synoikis-like hepatoma cells was detected by TEM, and the "Cell eat cell" phenomenon was recorded in detail as Entosis.1.2 Biology features of Synoikis-like BEL7402 cells1.2.1 Cell proliferation, viability, cell cycle progression, and the apoptosis assayBEL7402 cells were seeded in 96 well plate with or without polyHEMA as detached or attached cells as describe before. Cell viability and proliferation assays were performed by using Cell Counting Kit-8 (CCK8) according to the manufacture' s protocol. Cell cycle distributions of the detached and attached cells were determined by propidium iodide (PI) staining followed by flow cytometry (FCM). Subsequent histograms were analyzed by Multicycle software. Apoptosis statuses of the attached and detached cells were assayed for the amount of nucleosomal fragments in the cytoplasm by a Cell Death Detection ELISA kit, according to the manufacturer's instructions. The Synoikis-like cells were seeded on the suitable plate and their reattachments were recorded.1. 2. 2 Responses of Synoikis-like BEL7402 cells to chemotherapy, apoptosis-indueing ligand treatment and UV radiationTo determine whether deprival of attachment will influence the sensitivity to extracellular stimulators, the responses of attached and detached cells to chemotherapy and apoptosis inducing ligand were analyzed. Attached and detached BEL7402 cells were cultured in 96 well plate for 24 hours followed by treatment with 24μg/μl, 12μg/μl, 6μg/μl, 3μg/μl, 1.5μg/μl of 5 Flurouracil (5-FU) or 0. 2ng/ml, 2ng/ml or 20ng/ml of TNF related apoptosis inducing ligand (TRAIL) for 12 hours. The untreated cells were cultured under the same condition as control. Cell viability of the detached and attached cells with or without 5-FU or TRAIL treatment was determined by CCK8 kit. For UV radiation experiment, the hepatoma cells were seeded on the 96-well plate and cultured in RMPI1640 with 10% FCS for 24h, followed by exposing to UV radiation for 1h, 2h or 3h. The cell viabilities were determined by CCK8 kit.2 Molecular mechanisms of Synoikis-like cells2.1 Expression profile of Synoikis-like cells assayed by cDNA microarrayHuman oligonucleotide probe arrays were applied for analysis of mRNA expression levels corresponding to 22,000 transcripts. cDNA samples from attached and detached mature phase aggregations were labeled with Cy3 and Cy5 fluorescent dyes, respectively. DNA microarray hybridization and scanning was performed by Biocapital Campany. Expression profile of detached and attached hepatoma cells were analyzed and compared. MAS2.0 and Genespring7. 2 software were used to mining the data. The different expressed genes were classified. From the significantly different expressed genes, eight genes were selected to further identification.2.2 Survival mechanisms of Synoikis-like cells.The expression of BDNF/TrkB was detected by RT-PCR, real-time PCR and FCM. Synoikis-like cells viability was detected when extrinsic 10ng/ml BDNF was added.2.3 Synoikis-like cells apoptosis and its regulatory mechanisms.2.3.1 Changes of apoptosis signal pathway in Synoikis-like cellsThe expression of FasL/Fas and TRAIL/TRAIL-R were detected by RT-PCR, real-time PCR and FCM. The expression of XIAP and FLIP were detected by western.2.3.2 Construction of pSilencerBCL2L2 and Down-regulation of BCL2L2 reverses chemotherapy resistance of Synoikis-like cellsWe constructed a pSilencer329 siRNA vector to allow convenient ligation and established a PCR based method to select of pSilencer-BCL2L2. Inhibition of BCL2L2 expression by pSilencer-BCL2L was determined by real-time PCR, Viability of Synoikis-like cells after BCL2L2 inhibition and sensitivity to 5Fu were detected. The cells in either adherent or suspension conditions were triplicate treated with 1μM of wortmannin or 50μM of PD98059 for 12, 24, 36 or 48 hours followed by determining the cell viability by CCK8 kit. The PI3K/Akt inhibitor wortmaninn was used to study the regulatory mechanism of BCL2L2.RESULTS1 Establishment of a model and Synoikis-like hepatoma cells biology features1.1 Establish a model and morphological changes of hepatoma cells1.1.1 Establish a model to culture hepatoma cells in suspensionAttached and detached BEL7402 cells were generated by sequential cycles of culture on untreated (adhered) and polyHEMA treated cell culture wells (suspended).1.1.2 Three phases division after the detachment of hepatoma cellsThe hepatoma cells spontaneously self-assembled into three phases of aggregations after detachment according to their biological features, which is premature, mature and postmature phase. Mature phase cells are the most robust cells and might have the highest metastasis potential. Synoikis-like hepatoma cells were found in the mature phase and is the most interested ones we investigated in much details.1.1.3 Morphological change of hepatoma cellsWhen the cells were plated on polyHEMA coated plates, they gradually formed aggregates, beginning with single cells that subsequently rounded up and formed small aggregates of a few cells, collected into large, irregular clumps of cells, and condensed into spherical multiple cell aggregates around 24 h. The detached cells did not have any detectable proliferation or apoptosis. We termed them Synoikis-like hepatoma cells.The morphology of the suspended hepatoma cells changed significantly compared with their attached counterparts. Synoikis-like hepatoma cells could resist to EGTA induced calcium deprivation. Hepatoma cells in the aggregation were deeply compressed and resist to calcium deprived environment, the nuclear heterogeneity was reduced but the nuclear-cytoplasmic ratio was increased, and no obvious apoptosis was observed in Synoikis-like hepatoma cells. Entosis was found in the Synoikis-like hepatoma cells. Entosis was characterized as "cell eat cell" , which indicated there were some other mechanisms in the procession of Synoikis-like hepatoma cells anoikis resistance.1.2 Cell proliferation, viability, cell cycle progression, and the apoptosis assayThe Synoikis-like cells did not have any detectable proliferation or apoptosis as determined by CCK8 growth kit or apoptotic ELISA assay. Compared with attached controls, detached cells had an increased G0/G1 population as determined by FACScan which indicated these Synoikis- like BEL7402 cells was arrested in G0/G1 phase1.3 Responses of Synoikis-like BEL7402 cells to chemotherapy, apoptosis-inducing ligand treatment and UV radiationThe viability of the attached and detached cells after 12 h of 5-Fu or TRAIL treatment was assayed by the CCK8 kit. The viability of the 5-Fu-treated attached cells was decreased significantly compared with the untreated control cells at multiple dosages, including 1.5, 3, 6, 12, and 24μg/μl (P<0.05). However, for the suspended Synoikis-like cells, there was no significant difference between the treated and untreated cells, which indicates greater resistance of these Synoikis-like cells to chemotherapy, compared to their attached counterparts. The data derived from TRAIL treatment or UV radiation showed that sensitivity to TRAIL or UV-induced cell death was reduced greatly in detached cells compared with their attached counterparts(P<0.05).2 Molecular mechanisms of Synoikis-like cells2.1 Expression profile of Synoikis-like cells assayed by cDNA microarrayThe results of microarray analysis were derived by applying statistical methodology in order to determine general patterns of similarities and differences in gene expression between Synoikis-like cells and attached control groups. The microarray analysis showed there are significant differences in several aspects and these microarray data are consistent with the biological properties of Synoikis-like cells. 2.2 Survival mechanisms in Synoikis-like cellsBDNF/TrkB pathway was up-regulated in Synoikis-like cells. When extrinsic BDNF was added, the Synoikis-like cells exhibited higher proliferate index, which indicated that the BDNF/TrkB pathway was functional in Synoikis-like cells.2.3 Apoptosis and its regulatory mechanisms in Synoikis-like cellsThe mRNA detection of apoptosis molecules and FLIP, XIAP were not significantly influenced by Synoikis-like survival style, but FLIP, XIAP were increased at the protein level, which indicated apoptosis regulatory pathway were activated in Synoikis-like hepatoma cells.The expression of BCL2L2 was significantly decreased in the SiRNA transfected cells compared with nonsense transfected control. The inhibition rate was calculated to be 66%. Small interfering RNA-mediated depletion of BCL2L2 did not alter hepatoma multicellular aggregations' anoikis resistance, but reverse 5Fu resistance of hepatoma multicellular aggregations. We evaluated the response of attached and detached BEL7402 cells to the PI3K/AKT and ERK pathway inhibition, and found growth of attached BEL7402 cells was greatly inhibited under the effect of PI3K/AKT or ERK inhibition. However, the detached BEL7402 cells were much more resistant to PI3K/AKT or ERK inhibition. The expression of BCL2L2 was regulated by PI3K/Akt pathway; PI3K/Akt inhibitor could down regulate the expression of BCL2L2 in Synoikis-like hepatoma cells.CONCLUSIONSWe successfully built up a model in which we cultured hepatoma cells in suspension. Our result suggested that the hepatoma cells could resist to anoikis through a novel Synoikis-like survival style which may contribute to resistance to cancer treatment and facilitate metastasis. Determination of the key molecules responsible for this process may offer a good marker, as well as a target, for the metastasizing cells. We studied the mechanisms of Synoikis-like hepatoma cells. Our results implicate BCL2L2 as a regulator of drug resistance in hepatoma aggregations and suggest that targeted therapy against BCL2L2 might improve management the chemoresistance of hepatocarcinoma in conjunction with conventional anticancer therapy. The study of molecular mechanism of Synoikis-like will provide new approach to inhibit hepatoma cells metastasis.INNOVATIONS AND SIGNIFICANCES:1) We found that hepatoma cells resist to anoikis through Synoikis-like survival style and described the biology feature of Synoikis-like survival style for the first time.2) We studied the molecular mechanism of Synoikis-like survival style, and verified the key role of PI3K/Akt and MAPK/Erk pathway, BDNF/TrkB pathway in Synoikis-like hepatoma cells.3) By screen the results of expression profile of Synoikis-like hepatoma cells, we focus on 8 differently expressed genes, and we have constructed pSilencerBCL2L2 to produce siRNA and effectively inhibited the expression of these genes in Synoikis-like hepatoma cells. Our data indicated that BCL2L2 play an important role in Synoikis-like hepatoma cells.4) By study on sensitivity of Synoikis-like hepatoma cells to extracellular stimuli, we identified TrkB and BCL2L2 as targets for conquering metastasis of hepatoma cells. Synoikis-like survival style might shade light on the molecular anoikis resistance mechanism of hepatoma cells and help us develop new therapies that may alter metastasis potential of the hepatoma cells.